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Fungal DNA extraction from wheat


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#1 leonine

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Posted 22 April 2010 - 03:15 AM

Hi,
I have a FastPrep homogenizer in my lab. I use the lysing matrix (bead crusher) to lyse the cells. I get a high nanodrop (in the order of >300 ng/uL), but no fungal DNA is detected in my PCR system. This is fungi inoculated wheat.The primers are also fine, as +ve controls work.
I believe there is no fungal DNA that gets extracted. Is there a protocol to get preferentially fungal DNA out of the matrix?
please help! :D

#2 gebirgsziege

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Posted 22 April 2010 - 04:04 AM

usually the fast prep is good to get fungal DNA out of plant material. I once tried it and it works better than the ball mill we are using in our lab.

Which purification steps are you using after homogenisation?
Which fungi do you want to detect, as some like Fusarium species tend to make some trouble in DNA extraction.
Which primers are you using (ITS or a specific gen)?
So if you give me more details about your protocol I hope to be able to help you.


Ah and last but very important: have you tried to dilute your DNA-extract? You seem to have a very high DNA concentration which can lead to thermodynamic processes inhibiting PCR.
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#3 Adrian K

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Posted 22 April 2010 - 08:45 AM

Just curious, why don't you subculture your fungus onto agar media before you do dna extraction?
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#4 leonine

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Posted 22 April 2010 - 01:17 PM

usually the fast prep is good to get fungal DNA out of plant material. I once tried it and it works better than the ball mill we are using in our lab.

Which purification steps are you using after homogenisation?
One of the objectives for my research is to compare commercially available kits. I follow lysis and protein removal (proteinase K) step using the FastPrep protocol, and then follow the protocol from Qiagen Plant and Qiagen blood ( I will ultimately compare th results)
Which fungi do you want to detect, as some like Fusarium species tend to make some trouble in DNA extraction.
Murphy''s law... its Fusarium!! Ihave 5 species, F. avenaceum, F. culmorum, F. graminearum. F. poae and F. sporotrichioides.
Which primers are you using (ITS or a specific gen)?
I am using real time primers from a study by Nicholaise et al. i use SYBR green based detection.
So if you give me more details about your protocol I hope to be able to help you.


Ah and last but very important: have you tried to dilute your DNA-extract? You seem to have a very high DNA concentration which can lead to thermodynamic processes inhibiting PCR.
I will try that. But its unlikely that it is the Fusarium DNA in there.... Real time is really sensitive. I believe, somehow only the wheat is getting extracted. I ran a gel of the DNA before PCR, and I didnt see any smears/bands. thats whats making me wonder.



#5 leonine

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Posted 22 April 2010 - 01:20 PM

Just curious, why don't you subculture your fungus onto agar media before you do dna extraction?

I need to do it from the wheat to show the farmers-field application. (at least thats what my advisor says. Its hard for me to imagine a plough carrying farmer, holding a microcentrifuge tube!)

#6 gebirgsziege

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Posted 22 April 2010 - 11:42 PM

So ok. If you have to compare the commercial kits it is making trouble shooting more difficult, as you cannot change components of the protocol. But I know from my own work that the Quiagen Plant Kit usually works fine for detecting fungi from plant material, even if the fungi are there only in small amounts and the plants contain high amounts of PCR-inhibbiting substances. But knowing the Quiagen Plant Kit I think this should not be the problem, theoretically it should work, for Fusarium as well. The problems mentioned with Fusarium is that it produces a lot of polysaccharides (the reason for the "slimy" conidia) that tend to inhibit PCR. Those can be removed with a NH4Acetate step. So if the problem REALLY is the DNA (what I do not think), you could try to include a extra step with NH4Ac in your protocol.

To check the presence of fungal DNA I would advice you to make a pcr (standard or qPCR) with fungal AND plant (Eukaryote) primers. If these do not work, it really might be some inhibiton from the plant material or some other mistake with the DNA. At least the eukaryote primers should be positive with the plant material

You can also check the DNA extraction process by adding some fungal material to the plant material before starting with the extraction. If your samples are still negative, you should think about changes in the extraction process.

Have you tried to artificially inoculate wheat plants in the lab with the 5 species mentioned or do you only have plants from the field where you should detect the differen Fusarium species?

Ah and I think your supervisor is not expecting farmers to play around with pcr....DNA-based methods are much quicker than methods based on culturing and determinig the fungi (which is especially difficult in Fusarium, all species look very similar). Farmers need quick answers whethter or not they should use fungicides or they should grow something else on the field.

Edited by gebirgsziege, 22 April 2010 - 11:44 PM.

A man cannot be too careful in the choice of his enemies. (Oscar Wilde)




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